WO2003065986A2 - FcϵRI-BEARING HUMAN MAST CELL LINES - Google Patents

Abstract

Established human mast cell lines are disclosed wherein the cells express a functional FcϵRI receptor. In one embodiment, the cells exhibit stem cell factor (SCF) enhanced cell growth. In another embodiment, the cells express CD64. Methods of using these cells are also described.

Description

FcεRI-BEARING HUMAN MAST CELL LINES

PROIRITY CLAIM This application claims the benefit of U.S. Provisional Application No.

60/354,440, filed February 4, 2002, which is incorporated by reference in its entirety.

FIELD OF THE INVENTION This invention relates to the fields of allergy and immunology, more specifically to mast cell lines.

BACKGROUND

The mast cell is the major effector cell in the initiation of allergic inflammation. In addition, mast cells have been implicated in anaphylaxis. Changes in mast cell numbers accompanied by secretion of significant amounts of pro- inflammatory mediators also have been observed in delayed hypersensrtivity reactions, fibrosis, autoimmune disorders, and neoplasia. Studies in animal models of bacterial infection also suggest that mast cells have a protective role in host defense against pathogens in innate immunity along with a role in acquired immunity against parasitic infections. The satisfactory treatment of diseases, including asthma, continues to be a significant health care problem. Mediator release via FcεRI crosslinking on mast cells is known to be the most important means by which mast cells induce inflammation.

In addition, there is a condition termed "mastocytosis" that is a mast cell disease characterized by excessive numbers of mast cells in the skin, bone marrow, and internal organs. These patients may present with associated hematologic abnormalities such as a myelodysplastic syndrome or a myeloproliferative disorder. Mast cells are strategically located at the host environment interface and can provide an early defense against an invading pathogen. Primary mast cells have been isolated from tissues in order to study mast cells and the factors that they secrete. When activated, these mast cells have been shown to synthesize and release key immunoregulatory cytokines, one consequence of which is a rapid and vigorous immune response. In addition, an array of adhesion and immune receptors has been shown to be expressed on these mast cells. However, as the cells are primary, they cannot be propagated in vitro indefinitely.

The development of mast cells has also been characterized. Human mast cells originate from CD34+ progenitor cells when primary progenitor cultures are maintained in stem cell factor (SCF). However, these stem cell cultures yield a mixed population of cells that include other hematopoietic lineages. There currently is only one human mast cell line (HMC-1) in existence. However, the usefulness of this cell line is significantly limited by its lack of various cell surface receptors, including the FcεRI receptor. Clearly, a purified population of human mast cells that can be propagated in vitro and express the FcεRI receptor would facilitate studies of mast cells and their role in allergy and host defense.

SUMMARY Established human mast cell lines are disclosed. These cells express a functional FcεRI receptor. In one embodiment, a cell from the cell line, or a progeny of the cell, exhibits stem cell factor (SCF) enhanced cell growth. In another embodiment, a cell from the cell line, or a progeny of the cell, expresses CD64. Methods of using these cells, such as in screening agents that affect human mast cells, are disclosed herein.

The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description of several embodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1 is a series of bar graphs showing the effects of various cytokines and dose-response relationships of rhSCF on LAD1 mast cells. Fig. 1A shows mast cells cultured over 6 weeks in the presence of 100 ng/ml rhSCF alone or in combination with rhIL-3, rhIL-4, rhIL-5, or rhIL-6. Fig. IB shows the dose- response of 100, 500 and 1000 ng/ml of rhSCF alone or in combination with 100 ng/ml SDF-lα, and Fig. 1C shows the demonstration of contact inhibition at cell densities greater than 0.5 x 10⁶ cells/ml. Results are shown as the mean + SEM of 3 separate experiments.

Fig. 2 is a set of digital images of light microscopy of LADl cultures grown in the presence of (Fig. 2A) 100 ng/ml, (Fig. 2B) 500 ng/ml, and (Fig. 2C) 1000 ng/ml rhSCF (x 450).

Fig. 3 is a set of digital images showing light microscopy of LADl human mast cells. Fig. 3 A is Wright-Giemsa staining, Fig. 3B is acid toluidine blue staining, Fig. 3C is chloroacetate esterase staining, and Fig. 3D is tryptase staining. ( x 630).

Fig. 4 is a set of plots of FACS analyses of intracytoplasmic tryptase and chymase expression by LADl human mast cells. Upper panel - FSC by SS plot of LADl cells. Fig. 4A shows tryptase expression, Fig. 4B shows chymase expression, and Fig. 4C shows dual tryptase and chymase expression of approximately 37%. Isotype controls are shown as filled histograms. Results shown are representative of three experiments separately performed.

Fig. 5 is a digital image of electron microscopy of LADl human mast cells.

Ultrastructure examination shows a homogeneous population of mast cells with surface projections (Fig. 5A); multiple cytoplasmic granules with scroll patterns, lattice formation, dense cores, and homogeneous electron dense material (Fig. 5B) (x 10,000).

Fig. 6 is a FACS histogram analyses of CD surface markers. The horizontal axis represents CD marker analyzed, vertical axis represents total mast cell numbers analyzed. Fig. 6 A shows the number of cells expressing FcεRI. Fig. 6B shows the number of cells expressing CD22. Fig. 6C shows the number of cells expressing CD56. Fig. 6D shows the number of cells expressing CXCR4. Fig. 6E shows the number of cells expressing CD117. Fig. 6F shows the number of cells expressing CD56. Fig. 6G shows the number of cells expressing CD4. Fig. 6H shows the number of cells expressing CD35. Fig. 61 shows the number of cells expressing CD124. Fig. 6J shows the number of cells expressing CD9. Fig. 6K shows the number of cells expressing CD37. Fig. 6L shows the number of cells expressing CD132. Fig. 6M shows the number of cells expressing CD13. Fig. 6N shows the number of cells expressing CD45. Fig. 60 shows the number of cells expressing Glycophorin. LADl cells strongly express CD 4, 9, 13, 45, 117, 132, FcεRI and CXCR4, and to a lessor degree express CD22, CD31, CD35, CD37, CD56, CD 124, and glycophorin. Results shown are representative of three experiments separately performed.

Fig. 7 is a set of graphs showing dose-response for FcεRI-induced activation, upregulation of FcγRI by IFN-γ and activation, and β-hexosaminidase release from LADl human mast cells. Fig. 7 A shows the results when LADl cells were incubated at 37°C overnight with various concentrations of NP-IgE followed by incubation with 1 ng/ml NP-BSA. Results are expressed as % of β-hexosaminidase release of the total cellular content. Basal release was 2 - 3%. Each data point is the mean ± S.E. of n = 3 experiments performed in duplicate. Where error bars are not shown they are smaller than the symbol. Fig. 7B shows the results obtained when LADl cells were incubated at 37°C overnight with 1 μg/ml of IgE-NP followed by incubation with various concentrations of NP-BSA. Results are expressed as % of β-hexosaminidase release of the total cellular content. Basal release was 2 - 3%. Each data point is the mean ± S.E. of n = 3 experiments performed in duplicate. Fig. 7C is a FACS analysis of CD64 expression on naive and IFN-γ treated (15 ng) LADl cells in complete SFM for 48 hours, with unstained control. Fig. 7D shows the results obtained when LADl cells were pre-incubated with or without 15 ng of IFN-γ in complete SFM for 48 hours followed by activation with 1 μg/ml of monomeric F(ab')₂ fragments of anti-FcγRI IgG or mouse F(ab')₂ fragments of IgG. Results are expressed as % of β-hexosaminidase release of the total cellular content from naive and IFN-γ stimulated cells. Basal β-hexosaminidase release was 2 - 3%. Each data point is the mean ± S.E. of n = 3 experiments performed in duplicate. DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

I. Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology may be found in

Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19- 854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

The singular terms "a", "an", and "the" include plural referents unless context clearly indicates otherwise. The word "comprises" means "includes," hence "comprising A or B" means including A, B, or both A and B, unless context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including any provided explanations of terms, will control. In addition, the materials, methods, and examples are only illustrations and are not intended to be limiting.

CD4: Cluster of differentiation factor 4 polypeptide, a T-cell surface protein that mediates interaction with the MHC class II molecule. CD4 also serves as the primary receptor site for HIV on T-cells during HIV infection. The known sequence of the CD4 precursor has a hydrophobic signal peptide, an extracelluar region of approximately 370 amino acids, a highly hydrophobic stretch with significant identity to the membrane-spanning domain of the class π MHC beta chain, and a highly charged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985). CD64: A 72 kDa glycoprotein that is a cell surface antigen that is identical with the human Fc receptor for IgG (FcγRI). It is a subtype of receptor that binds monmeric ligands of IgG. The receptor is expressed on cells of the immune system, such as mononuclear phagocytes, amongst others. CD64 plays a key role in antibody dependent cytotoxicity and clearance of immune complexes. It is also known to contribute to the pathogenesis of immune-complex and autoimmune disorders. CD64 can be released from cells and can circulate in the body fluids as DBF (immunoglobulin binding factor) (Okayama et. al., J. Immunol. 164:4332, 2000).

Effective amount or Therapeutically effective amount: The amount of agent is sufficient to prevent, treat, reduce and/or ameliorate the symptoms and/or underlying causes of any of a disorder or disease. In one embodiment, an "effective amount" is sufficient to reduce or eliminate a symptom of a disease, or inhibit a pathological mechanism of the disease. In another embodiment, an effective amount is an amount sufficient to overcome the disease itself.

A "therapeutically effective amount" is a quantity of an agent sufficient to achieve a desired therapeutic effect in a subject being treated. For instance, in a cell or subject infected with HIV this can be the amount necessary to inhibit viral replication or to measurably alter outward signs or symptoms of the viral infection, such as an increase in T-cell counts. An example of such an effect is an amount of an agent that affects mast cells is an amount sufficient to measurably affect a property of mast cells and provide a beneficial physiological effect. When administered to a subject, a dosage will generally be used that will achieve target tissue concentrations (for example, in mast cells) that has been shown to achieve in vitro the desired biological effect.

Established cell line: A clonal population of cells that can be propagated in vitro for an extended period of time without losing a defining characteristic of the cell population. An "immortalized" mast cell line is a clonal population of mast cells that are established and can be propagated in vitro indefinitely. In one embodiment, the mast cell line is a human mast cell line. An established or immortalized mast cell line includes an established or immortalized population of mast cells that expresses the FcεRI receptor, and expresses CD64, and exhibits stem cell factor enhanced growth. An established or immortalized mast cell line also includes an established or immortalized population of mast cells that has two or more of the following characteristics (1) expression of the FcεRI receptor, (2) expression of CD64 and (3) stem cell factor enhanced growth. Thus, an established or immortalized mast cell that expresses the FcεRI receptor, expresses CD64 and exhibits stem cell factor enhanced growth is a cell of an established or immortalized mast cell line. Cells of an established or immortalized mast cell line include cells that have one or more genetic or phenotypic changes that do not affect ιa characteristic or a function of the mast cell. These cells are termed "mutants" derived from the mast cell line. One specific, non-limiting example of a mutant derived from a mast cell line is a mast cell that expresses functional FcεRI receptor, CD64, and/or exhibits stem cell factor enhanced growth, and includes a heterologous nucleic acid. One specific, non-limiting example of a heterologous nucleic acid is a nucleic acid that encodes an oncogene, such as a mutated ras gene or a myc gene. Another specific, non-limiting is a nucleic acid that confers resistance or sensitivity to an antibiotic. A third specific, non-limiting example is a nucleic acid that encodes a marker, such as beta-galactosidase or green fluorescent protein (GFP). Thus, one specific, non- limiting example of a mutant derived from an immortalized human mast cell line (e.g. LADl) is an immortalized mast cell that expresses functional FcεRI receptor and CD64, and exhibits stem cell factor enhanced growth and includes a mutated oncogene, such as ras gene. Another specific, non-limiting example of a mutant derived from an immortalized human mast cell line is an immortalized mast cell (e.g. LAD2) that expresses functional FcεRI receptor and CD64, and exhibits stem cell factor enhanced growth and expresses a heterologous nucleic acid encoding a cell surface marker. Yet another specific, non-limiting example of a mutant derived from an immortalized human a mast cell line is an immortalized mast cell (e.g. LAD2) that expresses functional FcεRI receptor and is transfected with a viral vector.

Expand: A process by which the number or amount of cells in a cell culture is increased due to cell division. Similarly, the terms "expansion" or "expanded" refers to this process. The terms "proliferate," "proliferation" or "proliferated" may be used interchangeably with the words "expand," "expansion," or "expanded." Fc receptor: Receptors that bind the Fc region of immunoglobulins and mediate immunoglobulin transport across membranes, stimulate a variety of cellular activities induced by antigen-antibody complexes, and possibly regulate the biosynthesis of antibodies. FcγRI specifically binds IgG. FcεRI specifically binds IgE. FcεRI is involved in the initiation of the allergic response. Binding of allergen to receptor-bound IgE leads to cell activation and the release of mediators, such as histamine, which are responsible for the manifestations of allergy. In one embodiment, the FcεRI receptor is a tetrameric complex, αβγ₂, which is found on the surface of mast cells, basophils, Langerhans cells, and related cells. Aggregation of IgE occupied FcεRI by antigen triggers both the release of preformed mediators such as histamine, as well as stimulation of the synthesis of leukotrienes. The receptor has been cloned and characterized (see U.S. Patent No.5,807,988). i one embodiment, the receptor is expressed on a mast cell. A "functional FcεRI receptor," when expressed on a mast cell, binds IgE. In one embodiment, a function of FcεRI binds IgE and causes release of β-hexosamidase from the mast cell. Gpl20: The envelope protein from Human Immunodeficiency Virus (HIV).

The envelope protein is initially synthesized as a longer precursor protein of 845- 870 amino acids in size, designated gpl60. Gpl60 forms a homotrimer and undergoes glycosylation within the Golgi apparatus. It is then cleaved by a cellular protease into gpl20 and gp41. Gp41 contains a transmembrane domain and remains in a trimeric configuration; it interacts with gp 120 in a non-covalent manner. Gpl20 contains most of the external, surface-exposed, domains of the envelope glycoprotein complex, and it is gpl20 which binds both to the cellular CD4 receptor and to the cellular chemokine receptors (e.g. CCR5)

The gpl20 core has a unique molecular structure, that includes two domains: an "inner" domain (which faces gp41) and an "outer" domain (which is mostly exposed on the surface of the oligomeric envelope glycoprotein complex). The two gpl20 domains are separated by a "bridging sheet" that is not part of either domain. Binding to CD4 causes a conformational change in gpl20 which exposes the bridging sheet and may move the inner and outer domains relative to each other. The CD4-binding pocket within gpl20 includes a number of residues which interact directly with Phe43 of CD4. The most important of these are Glu370, Trp427 and Asp368 (the latter residue also forms a salt bridge with Arg59 of CD4). These three residues are conserved in all primate lentiviruses.

Growth factor: A substance that promotes cell growth, survival, and/or differentiation. Growth factors include molecules that function as growth stimulators (mitogens), molecules that function as growth inhibitors (e.g. negative growth factors) factors that stimulate cell migration, factors that function as chemotactic agents or inhibit cell migration or invasion of tumor cells, factors that modulate differentiated functions of cells, factors involved in apoptosis, or factors that promote survival of cells without influencing growth and differentiation. Specific non-limiting examples of growth factors are bFGF and EGF. Heterologous: A heterologous sequence is a sequence that is not normally

(i.e. in the wild-type sequence) found adjacent to a second sequence. In one embodiment, the sequence is from a different genetic source, such as a virus or organism, than the second sequence.

Immimoglobulins: A class of proteins found in plasma and other body fluids that exhibits antibody activity and binds with other molecules with a high degree of specificity; divided into five classes (IgM, IgG, IgA, IgD, and IgE) on the basis of structure and biological activity. Immunoglobulins and certain variants thereof are known and many have been prepared in recombinant cell culture (e.g. see U.S. Patent No. 4,745,055; U.S. Patent No. 4,444,487; WO 88/03565; EP 256,654; EP 120,694; EP 125, 023; Faoulkner et al., Nature 298:286, 1982; Morrison, J. Immunol. 123:793, 1979; Morrison et al., Ann Rev. Immunol 2:239, 1984). A native (naturally occurring) immunoglobulin is made up of four polypeptide chains. There are two long chains, called the "heavy" or "H" chains which weigh between 50 and 75 kilodaltons and two short chains called "light" or "L" chains weighing in at 25 kilodaltons. They are linked together by what are called disulfide bonds to form a "Y" shape molecule. Each heavy chain and light chain can be divided into a variable region and a constant region. An Fc region includes the constant regions of the heavy and the light chains, but not the variable regions. Isolated: An "isolated" biological component (such as a nucleic acid molecule, protein or organelle) has been substantially separated or purified away from other biological components in the cell of the organism. Nucleic acids and proteins that have been "isolated" include nucleic components that naturally occur, i.e., other chromosomal and extra-chromosomal nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids. Lymphocytes: A type of white blood cell that is involved in the immune defenses of the body. There are two main types of lymphocytes: B-cells and T-cells.

Mast Cell: A tissue-based inflammatory cell of bone marrow origin that responds to danger signal of innate and acquired immunity with immediate and delayed release of inflammatory mediators (Taylor et. al., Allergy and Asthma Proc. 22:115, 2001). Mast cells have the capacity to rapidly release a wide variety of mediators, including, but not limited to, histamine, proteoglycans, proteases, and specific cytokines, permitting a rapid response following activation. In one embodiment, mast cells have the capacity to recognize a variety of infectious agents, including infectious organisms. Mast cells are known to express functional receptors. Specific, non-limiting examples of these receptors are FcεRI and Kit receptors. In one embodiment, mast cells express CD9, CD13, CD45, CD64, CD117, CD132, and CXCR4, amongst other markers. In another embodiment, the cells also express low levels of one or more of CD22, CD31, CD35, CD37, CD56, CD 124, and glycophorin. In yet another embodiment, a mast cell is enhanced by stem cell factor (SCF) or kit signal transduction for growth. "Enhanced" for growth refers to an increase in proliferation rate in response to SCF or kit signal transduction factor. In one embodiment, a cell is enhanced by SCF or kit signal transduction factor if the proliferation rate is increased more than 20%, more than 50%, or more than 100% in the presence of the factor, as compared to proliferation in the absence of the factor.

In a further embodiment, a mast cell expresses functional FcεRI receptors and is enhanced on SCF for growth. In another embodiment, a mast cell expresses functional the FcεRI receptors, expresses CD64, and is enhanced by SCF for growth. Mast cell proliferative disease: Diseases whose etiology lies in over production of mast cells. Representative examples include urticaria pigmentosa, mastocytoma, diffuse cutaneous mastocytosis, systemic mastocytosis, and mast cell leukemias. Nucleotide: "Nucleotide" includes, but is not limited to, a monomer that includes a base linked to a sugar, such as a pyrimidine, purine or synthetic analogs thereof, or a base linked to an amino acid, as in a peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotides joined by native phosphodiester bonds, between about 6 and about 300 nucleotides in length. An oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or DNA, and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can include linear sequences up to about 200 nucleotides in length, for example a sequence (such as

DNA or RNA) that is at least 6 bases, for example at least 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long, or from about 6 to about 50 bases, for example about 10-25 bases, such as 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Open reading frame: A series of nucleotide triplets (codons) coding for amino acids without any internal termination codons. These sequences are usually translatable into a peptide.

Pharmaceutical agent: A chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject or a cell. "Incubating" includes a sufficient amount of time for a drug to interact with a cell. "Contacting" includes incubating a drug in solid or in liquid form with a cell. An "anti-viral agent" or "anti- viral drug" is an agent that specifically inhibits a virus from replicating or infecting cells. Similarly, an "anti- retroviral agent" is an agent that specifically inhibits a retrovirus from replicating or infecting cells.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) of any length. Therefore, a polynucleotide includes oligonucleotides, and also gene sequences found in chromosomes.

Promoter: A promoter is an array of nucleic acid control sequences which direct transcription of a nucleic acid. A promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription. Protein or polypeptide: A biological molecule expressed by a gene and comprised of amino acids.

Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein preparation is one in which the protein referred to is more pure than the protein in its natural environment within a cell.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques.

Stem Cell Factor: A stromal cell-derived cytokine synthesized by fibroblasts and other cell types. SCF is a protein of 164 amino acids (RBL cells, rat) or 248 amino acids (human). The protein is extensively N-and O-glycosylated. Glycosylation is not required for biological activity since the non-glycosylated recombinant protein produced in E. coli is biologically fully active. The murine protein forms dimers. It displays approximately 80 percent sequence homology with the human factor. Proteolytic cleavage of a longer membrane-bound precursor protein of 220 and 248 amino acids, respectively, yields the active murine and human factor. The precursor also possesses biological activity. The receptor for SCF is the product of the oncogene designated kit, and is also known as CD117. SCF has many biological activities involving the cells of the hematopoietic lineage, including but not limited to lymphoid and myeloid cells. For example, in vitro SCF has been shown to be growth factor for primitive lymphoid and myeloid hematopoietic bone marrow progenitor cells. In addition, the subcutaneous administration of recombinant human SCF to baboons (Papio cynocephalus) or cynomolgus monkeys (Macaca fascicularis) leads to a pronounced expansion of mast cell populations in many anatomical sites, with expansion of numbers of mast cells in some organs by more than 100-fold.

Transfected: A transfected cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. As used herein, the term transfection encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, thereby producing a transfected host cell. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements known in the art. Viral vectors are recombinant DNA vectors having at least some nucleic acid sequences derived from one or more viruses.

Virus: Microscopic infectious organism that reproduces inside living cells. A virus consists essentially of a core of a single nucleic acid surrounded by a protein coat, and has the ability to replicate only inside a living cell. "Viral replication" is the production of additional virus by the occurrence of at least one viral life cycle. A virus may subvert the host cells' normal functions, causing the cell to behave in a manner determined by the virus. For example, a viral infection may result in a cell producing a cytokine, or responding to a cytokine, when the uninfected cell does not normally do so.

"Retroviruses" are RNA viruses wherein the viral genome is RNA. When a host cell is infected with a retrovirus, the genomic RNA is reverse transcribed into a DNA intermediate which is integrated very efficiently into the chromosomal DNA of infected cells. The integrated DNA intermediate is referred to as a provirus. The term "lentivirus" is used in its conventional sense to describe a genus of viruses containing reverse transcriptase. The lentiviruses include the "immunodeficiency viruses" which include human immunodeficiency virus (HTV) type 1 and type 2 (HrV-1 and HIV-2), simian immunodeficiency virus (SIN), and feline immunodeficiency virus (FIN).

HIN is a retrovirus that causes immunosuppression in humans (HTV disease), and leads to a disease complex known as the acquired immunodeficiency syndrome

(AIDS). "HIV disease" refers to a well-recognized constellation of signs and symptoms (including the development of opportunistic infections) in persons who are infected by an HTV virus, as determined by antibody or western blot studies.

Laboratory findings associated with this disease are a progressive decline in T- helper cells.

Cell lines

Disclosed herein are several immortalized mast cell lines that have the characteristics of primary human mast cells. In one embodiment, the mast cell line expresses a functional FcεRI receptor. In another embodiment, the mast cell line expresses CD64. In another embodiment, the mast cell line exhibits stem cell factor (SCF) enhanced growth, hi yet another embodiment, the mast cell line has two or more of the following characteristics (1) expression of a functional FcεRI receptor; (2) expression of CD64, and (3) SCF enhanced growth. Thus, in one embodiment, the mast cell line expresses functional FcεRI receptor, expresses CD64, and exhibits SCF enhanced growth. Specific, non-limiting examples of an established mast cell line include, but are not limited to, LADl (ATCC No. PTA-3908, deposited December 6, 2001), LAD2 (ATCC No. PTA-3909, deposited December 6, 2001), LAD3 (ATCC No. PTA-3910, deposited December 6, 2001), LAD4, and LAD5 (ATCC No. PTA-3997, deposited January 18, 2001) cells and their progeny. Also provided herein are "derivatives" of the described established mast cell lines. Such derivatives are further genetically altered by adding, for example, one or more genes for drug metabolizing enzymes, oncogenes, anti-oxidant agents, etc., thereby creating a continuous derivative of the cell line. Specific non-limiting examples of nucleic acids that are of use include, but are not limited to, Bcl-2, Bak, Bax, and c-kit Asp 816Val.

Established cell lines, including those provided herein, can be characterized, for instance, based on morphology (e.g., a cell diameter of about 8 to about to 15 microns with rough surfaces and cytoplasmic projections; or oval, round or multilobed nucleated cells with multiple cytoplasmic metachromatically staining granules) , cytochemistry (e.g., staining for CD64 expression; presence of functional FcεRI receptor), expression of cell markers (e.g., expression of tryptase or chymase), response to a growth factor (e.g., stem cell factor), or a disease marker (e.g., molecular defects associated with the relevant genetic disease). Known methods can be used to characterize the disclosed cell lines; examples of such methods are provided in the Examples described below.

The established cell lines described herein can be used, for example, where large numbers of homogenous (identical and cloned) cells would be useful, for instance in the study of factors that affect mast cells. The described cell lines can be used for both the identification and purification of growth factors important for growth and differentiation of human mast cells, as well as the characterization of how mast cells respond to such growth factors. In one embodiment, the mast cell lines disclosed herein can be utilized for screening compounds with agents that potentially may inhibit mast cell function, or other anti-inflammatory compounds. In another embodiment, the mast cell lines disclosed herein can be used to screen agents with the potential to inhibit mastocytosis, or other disorders of mast cells. Furthermore, nucleic acids encoding various polypeptide products can be introduced into the cells disclosed herein to evaluate the role of these polypeptides in mast cell function or differentiation. In yet another embodiment, the kit can be made that includes a container, such as a tube, vial, plate or polymeric bag that contains a mast cell obtained or derived from a mast cell line, or which contains a cell line phenotypically indistinguishable from the mast cell line. hi one embodiment, the mast cell line includes a c-kit Asp816Nal activating mutation, causing SCF-independent phosphorylation of CD117 (Furitsu et al., J. Clin Invest 92:1736, 1993), which gave rise to the notion that SCF independence and unlimited growth of HMC-1 cells in vitro might result from the Asp816Nal mutation. The mutation, however, has been demonstrated in mast cells obtained from essentially all patients with indolent mastocytosis, or mastocytosis with an associated hematologic disorder (Νagata et al., PNAS 92:10560, 1995; Longley et al., Proc Natl Acad Sci USA 96: 1609, 1999), and these mast cells do not have unlimited growth potential in vitro. Thus, this hypothesis has not been substantiated. Rather, it is now thought that the activating mutation along with host genetic polymorphisms, a second mutation, or chromosomal instability may be responsible for mast cell hyperplasia, at least in patients with mastocytosis (Metcalfe et al., Leukemia Research 25:577, 2001).

In one embodiment of the invention, a mast cell from a mast cell line described herein is transfected with a heterologous nucleic acid sequence. In one embodiment, the heterologous nucleic acid sequence encodes a polypeptide of interest. In one embodiment, the polypeptide of interest encodes any polypeptide or protein that is involved in the growth, development, metabolism, enzymatic or secretory pathways in a mast cell. Such polypeptides may be naturally occurring growth factors, cytokines, proteins, or enzymes, or may be fragments thereof. In another embodiment, the polypeptide encodes a marker. In yet another embodiment, the polypeptide is an enzyme involved in the conversion of a pro-drug to an active agent.

To transfect a mast cell with an exogenous nucleic acid, mast cells are cultured in vitro as described herein and an exogenous gene encoding the heterologous nucleic acid is introduced into the cells, for example, by transfection. The transfected cultured cells can then be studied in vitro or can be administered to a subject. The polypeptide encoded by the nucleic acid can be from the same species as the cell (homologous), or can be from a different species (heterologous). Thus, the described cell lines can be used to study malignant transformation by radiation, chemical, physical and/or viral agents, and transferred genes including oncogenes and high molecular weight genomic DNA from tumors, using standard assays. For example, a cloned viral oncogene N-ras is introduced into the mast cells, or mast cells including a c-kit nucleic acid encoding c-kit Asp816Val activating mutation. The nucleic acids are introduced using any means known to one of skill in the art, including, but not limited to, strontium phosphate, another transfection method, or electroporation. The subsequent ability of the newly transfected cells to proliferate is then assessed. Cells that are derived from the described established cell lines by the introduction of an oncogene(s) also can be used to screen for potential chemotherapeutic agents (for instance, using techniques described herein), especially those chemotherapeutic agents that may be specific for cells transformed by the activation of particular oncogenes or combination of oncogenes, or for the study of mastocytosis and agents to treat mastocytosis.

In one embodiment, the nucleic acid of interest encodes a polypeptide involved in growth regulation or neoplastic transformation of mast cells. Specific, non-limiting examples of nucleic acid sequences of interest are nucleic acids that encode oncogenes such as SV40 Tag, p53, myc, src, and bcl-2. In another embodiment, the nucleic acid sequence of interest encodes an enzyme. Specific, non-limiting examples of enzymes are proteins involved in the conversion of a pro- drug to a drug, cytochrome P450 enzymes, or viral thymidine kinase. In yet another embodiment, the nucleic acid sequence of interest encodes a transcriptional regulator. hi one embodiment, the nucleic acid sequence of interest is operably linked to a regulatory element, such as a transcriptional and/or translational regulatory element. Regulatory elements include elements such as a promoter, an initiation codon, a stop codon, mRNA stability regulatory elements, and a polyadenylation signal. A promoter can be a constitutive promoter or an inducible promoter. Specific non-limiting examples of promoters include the CMV promoter, an insulin promoter, and promoters including TET-responsive element for inducible expression of transgene. In another embodiment, the nucleic acid sequence of interest is inserted into a vector, such as an expression vector. Procedures for preparing expression vectors are known to those of skill in the art and can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989). Expression of the nucleic acid of interest occurs when the expression vector is introduced into an appropriate host cell. The vector can be a viral vector, such as an adenoviral or retroviral vector. One skilled in the art can readily identify vectors of use in eukaryotic cells.

Screening for Agents that Affect Mast Cells In one embodiment, the disclosed established cell lines are useful for screening agents that affect a property of a mast cell, such as mast cell proliferation, mast cell survival, receptor cross-linking, mediator release, signal transduction, or the secretion of a growth factor. Thus, an agent can be selected that stabilizes mast cells or affects the release of factors produced by mast cells. Agents of interest include, but are not limited to, chemical compounds, small molecules, polypeptides, and peptidomimetics. Specific, non-limiting examples of agents of interest include anti-inflammatory agents, growth factors, and cytokines.

The cell lines disclosed herein can be used for drug screening by growing the cells in vitro in medium containing the agent to be tested and contacting them with the agent of interest. After a suitable period of exposure, the effect of the agent is then determined. In one specific, non-limiting example, mast cell proliferation or survival is assessed. One of skill in the art can readily assess cell survival or proliferation, e.g., by trypan blue exclusion assay or related assays (Paterson, Methods Enzymol. 58:141, 1979), or by growth assays (e.g., MacDonald et al., Exp. Cell. Res. 50:417, 1968), all of which are standard techniques well known in the art. The effect of the agent on the parameter of mast cells, such as proliferation or survival, can be compared to a control. One specific, non-limiting example of a control are cells grown in the same medium that are not contacted with the agent. Another, specific non-limiting example of a control is a standard value. In another embodiment, the disclosed cell lines are used for identification of chemical and biological agents that affect mast cell function, such as tryptase and chymase expression (see Example 1), cytokine production, phagocytosis, and chemotaxis. One specific, non-limiting embodiment, β-hexosaminidase release following cross-linking of FcεRI and FcγRI is measured (see Example 1). In examples of such methods, chemical and biological substances are screened for their ability to induce or affect a parameter of mast cell function including, but not limited to, receptor cross-linking, signal transduction, or mediator release, by adding them to the growth medium of the established cells. After a suitable period of time, the function of the mast cells is evaluated. As described above, in one embodiment, the effect of the agent on the mast cells can then be compared to a control. Tests for mast cell function are known in the art (e.g. tests for chemotaxis: Taylor et al. Blood 98:1195, 2001; phagocytosis: Malaviya et al. Immunol Rev 179:16, 2001; and cytokine production: Okayama et al. J. Immunol 164: 4332, 2000).

Screening for agents that affect HIV binding

Recent studies suggest that HIN-1 gpl20 interacts with IgE V(H)3(+) on the surface of human mast cells, acting as a viral immunoglobulin superantigen and inducing mediator release (Marone et al., IntArch Allergy Immunol 125:89, 2001; Li et al., Blood 97:3484, 2001). Thus, the availability of human mast cell lines can be used to study the interaction of gpl20 with mast cells, and to screen for agents that affect this interaction. The mast cells may be used to in assays to screen for new compounds that inhibit gpl20 binding to mast cells, inhibit viral entry, or inhibit HIN replication. In one specific, non-limiting example, a viral isolate or an isolated gpl20 is contacted with the mast cells in the presence of an agent of interest. The ability of the virus or the gpl20 to bind to the mast cells, or the ability of the virus to enter or replicate in the mast cells, is then assessed. Nirion entry into mast cells can be measured using a variety of assays, such as, but not limited to, a quantitative PCR assay (Chun et al., Nature 387, No. 6629:183, 1997). hi one embodiment, the ability of labeled isolated gpl20 to bind to mast cells is assessed in the presence and the absence of the agent of interest. Suitable labels include, but are not limited to, enzymatic, fluorescent, or radioactive labels. Agents of interest include, but are not limited to polypeptides, isolated biological materials, chemical compounds, pharmaceuticals, or peptidomimetics. hi one embodiment, the ability of the agent to inhibit gpl20 binding to mast cells, inhibit HIN entry, or inhibit HTN replication is compared to a control. Suitable controls include cells not contacted with the agent or standard values.

EXAMPLES

Example 1 Materials and Methods

The ability to study human mast cells has previously been limited by the absence of appropriate human mast cell lines. For the past 13 years, the only cell line available to researchers and bearing any resemblance to human mast cells has been the HMC-1 mast cell leukemic line (Butterfield et al., LeukRes 12:345, 1988), which has two major deficiencies which limit its usefulness. First, despite the presence of C-kit (CD 117) surface receptors, HMC-1 is a CD117 ligand (stem cell factor, SCF)-independent cell line. HMC-1 cells grow well in media with fetal serum in the absence of SCF, a known human mast cell growth factor (Kirshenbaum et al., J. Immunol. 146:1410, 1991), in contrast to normal human mast cells (Durand et al, Blood 84:3667, 1994; Kirshenbaum et al., Blood 94:2333, 1999). Second, HMC-1 cells possess non-functional FcεRI receptors due to an aberrant FcεRI α- subunit (Butterfield et al., LeukRes 12:345, 1988). Thus, many in vitro studies are conducted using pure human mast cells derived from CD34+ pluripotent progenitor cells. This approach is time-consuming, costly, requires at least 8-10 weeks for pure, albeit low numbers of pure mast cells to appear in culture, and likely has significant donor variability. Thus, the following methods were used to produce human mast cell lines:

LADl Culture. Human bone marrow (BM) from a patient with a severe form of mastocytosis was obtained and processed, following informed consent. Twenty ml of BM aspirates were collected for study. Mononuclear cells were collected following separation with lymphocyte separation medium (ICΝ Biomedicals, Aurora, OH). Cells were placed at a concentration of 5 x 10⁵ cells/ml in serum-free media (SFM) (StemPro-34; Life Technologies, Grand Island, ΝY) supplemented with 2 mM L-glutamine, 100 IU/ml penicillin, 50 μg/ml streptomycin (complete SFM), 100 ng/ml recombinant human stem cell factor (rhSCF), 100 ng/ml rhIL-6, and 30 ng/ml rhIL-3 (first wk only) (Peprotech, Rocky Hill, NJ). Hemidepletions were performed weekly with media containing 100 ng/ml rhSCF and 100 ng/ml rhIL-6. Following establishment of the LADl cell line, rhIL-6 was removed from cultures, and cells were maintained with complete SFM supplemented with 100 ng/ml rhSCF. In some experiments, cultures were supplemented 100 ng/ml rhSCF and either 30 ng/ml rhIL-3, rhIL-4 100 ng/ml, rhIL-5 10 ng/ml or rhlL- 6 100 ng/ml, with hemidepletions performed as above. In rhSCF dose-response experiments, cultures were supplemented with 100, 500 or 1000 ng/ml rhSCF alone or with 100 ng/ml SDF-lα. Cell aliquots were taken weekly for determination of total mast cell numbers, and for histochemical and immunohistochemical staining. Kimura's stain was used to stain mast cells on hemocytometers (Kimura et al., Clin Allergy 3:195-2000). Acidic toluidine blue (pH 1.0) was used to stain mast cell cytopreparations (Cytospin 3; Shandon, Pittsburgh, PA) or sections fixed in a mixture of 2% paraformaldehyde and 1.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3, following sectioning for electron microscopy. Wright- Giemsa, chloroacetate esterase staining and qualitative tryptase enzyme determinations on cytocentrifuged cell preparations were performed as described (Kirshenbaum et al., Exp Hematol 26:245, 1998).

Freezing and Thawing of LADl. LADl cells required special non-DMSO, non-FBS containing cryopreservative for optimal freezing and recovery. Briefly, cultured LADl cells were centrifuged at 1200 rpm for 10 minutes and aliquoted at 10 x 10⁶ cells per 1.5 ml of pZerve cryopreservative (Celox Laboratories, St Paul, MN) (1.5 ml of cell suspension per freezing tube). Cells were incubated for 30 minutes at 22°C, transferred to -20°C for 1 hour, followed by -70°C for 1 hour, and finally liquid nitrogen until needed. For thawing, aliquoted cells were warmed quickly, very gently resuspended and allowed to equilibrate in small flasks for 6 hours at 22°C to which an additional 1.5 ml of complete SFM supplemented with 1000 ng/ml rhSCF was added. Cells were gently agitated every 15-30 minutes to break up any cell clumping. Cell viability was assessed with either trypan blue staining or FACS analysis using PI. Flasks were then transferred to 37°C in 95% air/5% CO₂. Following 24 hours of incubation, an additional 3 ml of complete SFM with rhSCF was added, with hemidepletions and cell counts being performed as described above.

FACS Analysis. LADl human mast cells were analyzed for surface antigens as described (Kirshenbaum et al., Blood 94:2333, 1999). For each antigen studied, 2-5 x 10⁴ LADl cells were first incubated in 1 x PBS containing 0.1% BSA and 1% milk for 1 hour at 37°C, followed by staining with 1-3 ug/ml of fluorescein isocyanate (FITC), R-phycoerythrin (PE), PE Cyanine 5 (PECy5), or allophycocyanine (APC) -conjugated monoclonal antibodies for 30 minutes at 37°C. Surface antigens examined included CD2, CD3, CD4, CD5, CD8, CD9, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD31, CD32 CD34, CD35, CD37, CD41a, CD45, CD56, CD64, CD72, CD103, CD117, CD124, CD125, CD132, CCR3, CCR5, CXCR4 and glycophorin (Becton-Dickinson, San Jose, CA). Cells examined for Fc_εRI were incubated overnight at 37°C with 2 μg/ml FITC- conjugated human IgEps (see below). Cells were then washed and resuspended in cold PBS containing 0.1% BSA. Control cells were unstained or stained with an irrelevant mouse IgGi . Cell analysis was performed using a FACSCalibur (Becton Dickinson, San Jose, CA) and CellQuest software (Becton Dickinson).

FITC Conjugation ofIgEp$. IgEps was conjugated with FITC as previously described (Forni, Immunological Methods 8:151, 1979). Protein concentration (mg/ml) was calculated using the formula:

OD^go - 0.35 x ODao 1.4

Moles of FITC per mole of IgE were calculated using the formula:

2.87 x ODjos OD₂₈₀ - 0.35 x OD₄₉₅ Conjugated IgE_PS was 2.66 mg/ml with 7.3 moles FITC per mole IgEps.

Cell Activation and β-Hexosaminidase Release, β-hexosaminidase release of LADl human mast cells was measured following cross-linking of FcεRI and FcγRI (CD64) receptors, as previously described (Dastych et al., J. Immunol. 158:1803, 1997; Okayama et al., J. Immunol. 164:4332, 2000). For FcεRI-induced release, cells were sensitized by overnight incubation in complete SFM containing various concentrations of NP-specific IgE. Sensitized cells were rinsed with HEPES buffer (HEPES 10 mM, NaCl 137 mM, KCl 2.7 mM, sodium phosphate monobasic 0.4 mM, glucose 5.6 mM, calcium chloride 1.8 mM, magnesium sulfate 1.3 mM, pH 7.4) containing 0.04% BSA. Cells were centrifuged at 1200 rpm at 22°C for 10 minutes and resuspended in the same buffer. In all experiments, 5000 cells (90 μl) were placed in individual wells of 96-well flat-bottom microtiter plates (Falcon, Becton Dickinson, Franklin Lakes, NJ). The plates were warmed to 37°C in a hot air oven for 10 minutes. In dose-response experiments using various concentrations of NP-BSA, 10 μl of a 10X solution of NP-BSA (30:1) conjugate (Biosearch Technologies, Inc., Novoto, CA) was added for 30 minutes at 37°C. Plates were then centrifuged at 1200 rpm at 4°C for 5 minutes to pellet the cells, and 50 μl of each supernatant was removed for β-hexosaminidase assay. To determine the remaining cellular content of β-hexosaminidase, 150 μl of distilled water was added to each well and the plate was placed in a freezer at -20°C until the solutions in the wells were frozen. After thawing, the contents of each well were mixed by pipetting up and down, and a 50 μl aliquot was taken for the β-hexosaminidase assay. For FcγRI-dependent activation, LADl cells were pre-incubated with or without 15 ng interferon gamma (TFN-γ) in complete SFM for 48 hours. The expression of FcγRI on IFN-γ-treated cells was then confirmed by FACS analysis. Cells were rinsed with HEPES buffer containing 0.04% BSA, centrifuged at 1200 rpm at 22°C for 10 minutes and resuspended in the same buffer. 5000 cells (90 μl) were placed in individual wells of 96-well flat-bottom microtiter plates and warmed to 37°C in a hot air oven for 10 minutes. Individual wells were then incubated with 1 μg/ml of monomeric F(ab')₂ fragments of anti-FcγRI IgG or mouse F(ab')₂ anti-CD64 fragments (10 μg/ml; Jackson ImmunoResearch Labs, West Grove, PA) for 30 minutes at 37°C followed by centrifugation at 1200 rpm at 4°C for 5 minutes to pellet the cells. Supernatant and cellular β-hexosaminidase determinations were determined as for FcεRI-induced release. β-Hexosaminidase Assay, β-hexosaminidase was assessed in cell supernatants and lysates. Briefly, 50 μl of cell lysate or supernatant was placed in individual wells of 96-well flat-bottom microtiter plates, together with 100 μl of P- nitrophenyl-N-acetyl β-D glucosaminidase (PNAG) solution (2.5mM PNAG in 0.04 M citrate buffer; pH 4.6). The reaction was allowed to proceed for 90 minutes at 37°C, and then terminated by the addition 50 μl of 0.4 M glycine solution. The appearance of the colored product was measured using a Perkin Elmer HTS 7000 plate reader at 405 nm wavelength using a reference filter of 570 nm. After normalizing the data for the aliquot size and the media content remaining in the cellular fraction, degranulation was calculated as the % of total cellular content, at the beginning of the experiment, found in the media after cellular activation.

Electron Microscopy. LADl cells cultured in rhSCF were harvested and fixed for electron microscopy and immunohistochemistry (Kirshenbaum et al., Exp Hematol 26:245, 1998). Sections were examined using a Hitachi 7100 electron microscope.

Intracytoplasmic Staining for Tryptase and Chymase. LADl human mast cells were permeabilized and stained for tryptase and chymase as described (Kirshenbaum et al., Blood 94:2333, 1999; Prussin et al., J. Immunol Methods 182:115, 1995). Briefly, 50-100 x 10⁵ cells were fixed with 4% paraformaldehyde, washed and incubated with a blocking solution of 1 x PBS-Saponin (PBS-S) containing 0.1% BSA and 1% milk for 1 hour at 37°C. For tryptase staining only, cells were incubated with 3 μg/ml mouse anti-human tryptase (1.14 mg/ml)

(Chemicon, Temecula, CA) for 1 hour at 37°C, washed and incubated with 30 μg/ml PE-conjugated goat anti-mouse IgG (1 mg/ml; Southern Biotechnology, Birmingham, AL) for 30 minutes at 37°C. For chymase staining, cells first stained for tryptase were washed and incubated with 3 μg/ml biotinylated mouse anti-human chymase (3 mg/ml) (Chemicon) for 1 hour at 37°C, washed and incubated with 2 μg/ml allophycocyanine (APC)-conjugated streptavidin for 30 minutes at 37°C. Following staining, all cells were washed and resuspended in cold 1 x PBS containing 0.1% BSA. Control cells were unstained or stained with an irrelevant mouse IgGi. Cell analysis was performed using a FACSCalibur and CellQuest software.

IL-4-Induced Apoptosis of LADl Cells. Since TL-4 appeared to decrease LADl growth and proliferation in the presence of rhSCF, LADl cells were examined for IL-4 induced apoptosis and protection by IL-6 as described (Mekori et al, J Clin Immunol 21:171, 2001; Oskeritzian et al., J. ImmunolA63:5105, 1999). Briefly, 2 x 10⁵ LADl cells per ml were cultured for at least 48 hours in complete SFM supplemented with and without 100 ng/ml rhSCF and 100 ng/ml rhIL-6. In certain conditions, rhIL-4 at 100 ng/ml was added at time 0 with subsequent cell counts and viability by trypan blue exclusion being examined at 2, 4 and 7 days, and FACS analysis for apoptosis and necrosis being examined at 4, 24, 48 and 96 hours and 7 days. As outlined above, cells processed for FACS analysis were harvested, washed and incubated in cold PBS for 30 minutes. Cells were then incubated with 7-Amino-actinomycin D and/or PE-conjugated annexinV for 15 minutes at 4°C. Primary human mast cells deprived of rhSCF for 12-24 hours, and a biotinylated soy protein were used as a positive and negative apoptosis controls, respectively. Primary human mast cells fixed in 4% paraformaldehyde and resuspended in PBS/Saponin were used as a positive PI control. Cells were examined on a FACSCalibur using CellQuest software.

Telomerase Assay. Telomerase was assayed using a telomerase polymerase reaction/ELISA kit as described (Chaves-Dias et al., J. Immunol. 166:6647, 2001). Cells (3 x 10⁵) were washed twice in ice-cold PBS by centrifugation at 3000 x g for 10 minutes at 4°C. Pelleted cells were resuspended in 200 ul of lysis reagent as supplied in the kit and left on ice for 30 minutes. Cell lysates were centrifuged at 6000 x g for 20 minutes at 4°C, and the supernatant extracts were stored at -80°C until use. The telomeric repeat amplification protocol was performed with extracts containing 1500 cell-equivalents for each reaction (15-minutes incubation and 25 cycles). The hybridization and ELISA procedures were performed according to the manufacturer's protocols (Roche Molecular Biochemicals, Indianapolis, IN), and the absorbance of the final colored reaction product was measured in a microliter plate reader at 450 nm. Each arbitrary unit was defined as absorbance per 1500 cell equivalents.

Karyotypic analysis. To determine the karyotype of LAD 2, metaphases were harvested from 5-10 x 10⁶ cells that were arrested in metaphase using colcemid overnight, placed in hypotonic potassium chloride, and fixed in methanol and acetic acid. Analysis using spectralkaryotyping was performed for studying chromosomal aberrations including aneuploidy and translocations.

Example 2 Isolation and Characterization of LADl Mast Cells

Mononuclear cells isolated from a patient with a severe form of mastocytosis were observed, in the presence of 100 ng/ml rhSCF and complete SFM, to grow and proliferate (Fig. 1A). The addition of rhIL-3, rhIL-5, rhIL-6, or SDF-lα, the ligand for CXCR4, did not increase cell proliferation, h contrast, the addition of rhTL-4 reduced cell proliferation by at least 50% starting at 1 week and continuing through 6 weeks in culture (Fig. 1 A), raising the question whether rhIL-4 induces human mast cell apoptosis (see below). Dose-response experiments using complete SFM in combination with 100, 500 and 1000 ng/ml of rhSCF, with and without 100 ng/ml SDF-lα, showed a two-fold increase in cell proliferation by 2 weeks when cells were cultured in 500 or 1000 ng/ml rhSCF (Fig. IB). A number of cells appeared vacuolated when cultured in 100 ng/ml but not higher concentrations of rhSCF (Fig. 2). Cells appeared to demonstrate contact inhibition, despite weekly hemidepletions and replacement with complete SFM and SCF, when cells densities were greater than 0.5 x 10⁶ cells/ml (Fig. 1C). Viability in culture was measured regularly and always greater than 95%.

Wright-Giemsa staining (Fig. 3 A) showed oval, round or multilobed nucleated cells with multiple cytoplasmic metachromatically staining granules on acid toluidine blue staining (Fig. 3B). Granules stained positively for chloroacetate esterase (Fig. 3C) and for tryptase (Fig. 3D). Histochemical and immunohistochemical staining of LADl were therefore consistent with published accounts of human mast cells. FACS analysis of LADl cells for tryptase expression was consistent with histochemistry, showing >99% positivity (Fig. 4A). FACS analysis was chymase expression was less positive (Fig. 4B), with approximately 37.5% of LADl cells expressing both tryptase and chymase (Fig. 4C). Thus, FACS analysis (Fig. 4) showed that LADl cells are composed of two tryptase-containing mast cell phenotypes,: >99% of LADl cells are positive for tryptase only (MCx cells), and approximately 31.5% of LADl cells express both tryptase and chymase (MC_TC cells). No chymase only (MCc) cells were noted. Without being bound by theory, tryptase and/or chymase expression may depend on the state of maturity of individual cells

Ultrastructure of LADl cells was consistent with published accounts of human mast cells (Kirshenbaum et al., J. Immunol. 148:772, 1992; Kirshenbaum et al., J. Immunol. 146:1410, 1991). Cells measured 8-15 microns in diameter with rough surfaces and cytoplasmic projections. Numerous cytoplasmic granules of consistent size were noted with scroll, lattice, electron dense core and homogeneous patterns noted (Fig. 5). Immunogold labeling demonstrated the presence of tryptase within cytoplasmic granules (Fig. 5, inset). As seen on light microscopy, granules were better formed and cytoplasmic vacuolation was significantly reduced when cells were cultured with 1000 ng/ml rhSCF rather than 100 ng/ml.

To demonstrate the presence of CD surface markers, LADl and LAD2 cells were incubated with monoclonal antibodies for CD2, CD3, CD4, CD5, CD8, CD9, CD13, CD14, CD15, CD16, CD19, CD20, CD22, CD25, CD31, CD32, CD34, CD35, CD37, CD41a, CD45, CD56, CD64, CD72, CD103, CD117, CD124, CD125, CD132, CCR3, CCR5, CXCR4, glycophorin, and Fc_εRI. LAD cells strongly expressed surface FcεRI, CD 4, 9, 13, 45, 64, 71, 103, 117, 132 and CXCR4 markers, and to a lessor degree, CD14, CD22, CD31, CD32, and CCR5 (Fig. 6). The strong expression of surface CD4 for human mast cells was unexpected, and with the exception of an intermediate peripheral blood-derived metachromatic cell in atopic individuals (Li et al., Blood 97:3484, 2001), is previously unreported.

The expression of CD4 and CXCR4 is of interest, since HIN-l infection remains a major health problem. Recent reports suggest that peripheral blood metachromatic cells, most likely basophils, are HIN-1 susceptibility in allergic individuals due to their surface expression of CD4 and the chemokine receptors CCR3, CCR5, and CXCR4. Thus, LADl human mast cells can provide an unlimited resource and a new human mast cell model for the study of human mast cell HIN-1 susceptibility via CD4 and chemokine receptors. The effect of HIN-1 infection of LADl cells on SCF- enhanced growth and release of mediators via functional FcεRI receptors, and conversely, resistance of activated LADl mast cells to HIN-1 susceptibility can be examined by the provision of the mast cells disclosed herein. Similarly, the satisfactory treatment of allergic diseases including asthma continues to be a significant health care problem, and the mast cell is the major effector cell in the initiation of allergic inflammation. Human mast cells, in response to SDF-lα, transmigrate and produce IL-8, a neutrophil chemoattractant, without affecting TΝF-α, IL-lβ, JL-6, GM-CSF, IFΝ-γ or RAΝTES (Lin et al., J. Immunol. 165:211, 2000). Neutralizing antibodies to CXCR4, the only known receptor for SDF-lα, reduce lung eosinophilia and allergic airway inflammation (Gonzalo et al., J. Immunol. 165:499, 2000). Thus, LADl mast cells can be utilized as a new human mast cell model for studying the CXCR4/SDF-lα interaction, neutrophil and eosinophil chemotaxis, and asthmatic airway disease. As described herein, the cell lines disclosed can be used for the testing and development of pharmaceuticals that affect human mast cell FcεRI/IgE binding, human mast cell activation, or mediator release.

Example 3 Further Characterization of LADl Cells:

Functional Characterization and the Effect of Growth Factors

Experiments were performed to demonstrate functional FcεRI receptors, and thus release of β-hexosaminidase was measured. Dose-response experiments were designed for various concentrations of IgE-NP and NP-BSA. As shown, LADl cells released a mean of 40% β-hexosaminidase with 10 ng/ml NP-BSA (Fig. 7A) and 10 ug/ml IgE-NP (Fig. 7B), consistent with published accounts of CD34+-derived human mast cell β-hexosaminidase release. Background release was no higher than 3% in all experiments.

Human mast cells have been shown to upregulate CD64 (FcγRI) following incubation with IFN-γ, and release β-hexosaminidase with the same order of magmtude as with FcεRI cross-linking (Okayama et al., J. Immunol. 164:4332, 2000). LADl cells were thus incubated with IFN-γ for 48 hours and examined for CD64 expression. Naive LADl cells had low-level expression of CD64 which increased in the presence of IFN-γ (Fig. 7C). Percentage of β-hexosaminidase release increased almost 3.5-fold from 23% to 87% when naive cells were pre- incubated with TPN-γ (Fig. 7D).

As might be expected and has been reported for mature mast cells, the addition of rhIL-3, rhIL-5, rhIL-6 and SDF-lα did not influence LADl cell numbers. Recombinant hIL-4, in contrast to other cytokines studied herein, reduced mast cell numbers by at least 50% starting at 1 week and continuing through 6 weeks in culture (Fig. 1A), consistent with other reports (Oskeritzian et al., J.

Immunol. 163:5105, 1999). To detennine if IL-4 induced apoptosis of LADl cells, cells were cultured in complete SFM supplemented with rhSCF with and without rhIL-4. The addition of rhlL-4 to cultures supplemented with rhSCF alone or with rhIL-6 did not induce apoptosis or necrosis. Tumorigenic mast cell lines such as RBL-2H3 and HMC-1 express persistently high telomerase activity throughout the cell cycle (Chaves-Dias et al., J. Immunol. 166:6647, 2001). To determine if LADl cells contain tumorigenic telomerase activity, cell lysates were assayed by PCR/ELISA. LADl telomerase activity (arbitrary units) averaged 2200, comparable with 2000 for RBL-2H3 and 2300 for HMC-1 cells. As described above (see Example 1), karyotypic analysis using spectralkaryotyping of stimulated LAD 2 cells revealed a 48, XXY, +1, +5, -16 karyotype. Significant loss of material from chromosome 1 could be suggestive of-lp. Example 3 Isolation and Characterization of Additional Mast Cell Lines

LADl cells have tumorigenic levels of telomerase found in HMC-1 and other established cell lines, in addition to changes in chromosome number and G- banding, which might have given LADl cells unlimited growth potential. Despite these observations, in the absence of SCF, LADl cells survive in culture unlike primary mast cell cultures which under apoptosis and rapidly deteriorate in the absence of SCF. Thus, without being bound by theory, it is possible that at least two phenomena were needed for the clone of human mast cells and for these cells to have survived and proliferated in culture: the presence of tumorigenic levels of telomerase needed for cell survival, and the requirement for SCF binding with CD117 receptors for reasonable growth and proliferation. Without being bound by theory, these same two prerequisites may be required for immortalization and production of any additional human mast cell lines. Mature human mast cells, whether derived from progenitors present in bone marrow, peripheral blood, cord blood, or fetal liver, are known to survive and proliferate optimally when cultured in serum-free media supplemented with rhSCF, in addition to rhIL-3 and rhIL-6. This is the first description of a human mast cell line that resembles these in vitro systems, and has functional FcεRI receptors. Additional mast cell lines were also developed and characterized. These cell lines were called LAD2, LAD3, LAD4, and LAD5 (see Table 1).

TABLE 1. Additional human mast cell lines in development* Designation Tissue Source ATCC No.

LAD2 Bone Marrow PTA-3909

LAD3 Bone Marrow PTA-3910

LAD4 Bone Marrow to be determined

LAD5 Bone Marrow PTA-3997

*With the exception of cyto genetic studies, LADl data presented herein are representative of these mast cell lines. For the additional human mast cell lines noted above, 2-5 ml of bone marrow aspirate was obtained at different times from the posterior iliac crest (LAD 3-5) or sternum (LAD 2) of the same patient from whom LADl cells were obtained. Mononuclear cells were isolated by density gradient centrifugation. The cells were plated at a concentration of 1 x 10⁶ cells per ml in Stem-Pro serum-free media supplemented with 100 ng/ml rhSCF. Hemidepletions with media containing rhSCF were performed weekly. Studies were performed to characterize these cell. Thus, for LAD2, mediator release following FcεRI crosslinking and tyrosine phosphorylation was assessed. For LAD3, Wright-giemsa staining, tryptase staining, flow cytometry, response to growth factors, chromosomal analysis and mutational analysis have been performed.

Example 4 LAD Enzyme Release (LAD-DER) Assay

An assay, termed the LAD-DER assay, was developed to assay mast cell mediator (B-hexosaminidase or histamine) release using patient's serum and activated LAD cells. For this assay, as little as 5000 cells per well (96 well plate) are needed. Generally, a 96 well plate is utilized. For patients allergic to ragweed or bee venom, cells are incubated overnight with 1/50 to 1/2 (e.g. 1:50, 1:20, 1:10, 1:5, 1:2) dilutions of serum containing anti- ragweed or anti-venom IgE (as quantitated by RAST). The cells are washed and resuspended as 5000 cells per 90 microliters of buffer (e.g., Hepes with 0.04% BSA). Cells are plated and the FcεRI receptors are cross-linked by the addition of 10 microliters of buffer or lOx concentrations of ragweed IgE, whole ragweed extract or vespid venom.

Following incubation at 37° C, plates are spun and 50 microliters of supernatant is removed for each well to another plate. One hundred fifty microliters of distilled H 0 are added to remaining cells, and the plates are frozen at -80°C and then thawed. Fifty microliters of thawed cell extract are also removed to a new plate. One hundred microliters of PNAG are added to each new plate, with incubation at 37° C for 90 minutes. The reaction is stopped using 50 microliters of glycine (for yellow color development) and plates read at 405 nm.

The results demonstrate that this assay is sensitive to 0.15-0.3 ng/ml extract or AgE, respectively, and 0.3 ng/ml venom. Thus, this assay can be used to measure ability of drugs to block or potentiate allergen-induced release, or to determine safety of drugs or vaccines by not causing mast cell release, allergic reactions or anaphylaxis.

Example 5 Infection of LAD cells with HIV

Infection with T-trophic human immunodeficiency virus (HIV) requires the expression of the CD4 receptor and the CXCR4 co-receptor on cells, while infection with M-trophic HIN requires expression of CD4 and CCR5 on cells. Mature CD34⁺ -derived primary cultured mast cells cultured for 8 weeks in vitro lack CD4 and cannot be infected. In previous studies, early 4 week old mast cell progenitors have been documented to show infection with M-trophic virus only. However, new data suggests that in patients with very low CD4 counts and high viral burden of HIV, mature tissue mast cells may show infection with HIN. Thus, LAD cells provide a unique opportunity to study HIN infection in mast cells, and provide a system to screen for agents that prevent HIN infection of mast cells, or HIN replication in mast cells.

LAD cells show unique ability to become infected with HIN T-trophic (requires CD4 receptor and CXCR4 co-receptor) and M-trophic (requires CD4 and CCR5) viruses. Specifically, LAD cells express CD4, CXCR4 and CCR5 on the cell surface.

Thus, LAD cells which resemble mature mast cells can serve as model for tissue mast cells and HIN infection. A method is therefore provided to test anti- viral agents, by contacting LAD cells with the agent and HIN and assessing infection of the mast cells. Spreading of virus between mast cells can also be assessed by contacting LAD cells known to be infected with HIN with an agent of interest. A LAD cell not infected with HIN is then added to the culture system. It should be noted that the uninfected LAD cell can include a marker to identify these cells. The ability of the virus to infect the (previously) uninfected cell is assessed.

In addition, the safety of antiviral agents (such as an agent to treat or prevent infection with HIN) can be tested using the assays described herein. Specifically, LAD cells can be used to test that ability of an agent to treat or prevent HIN. LAD cells can then be used to test safety of agents using the LAD-ER assay disclosed herein. Specifically, an agent is selected that does not induce mast cell release of B- hexosaminidase or histamine is selected (using the LAD-DER assay, see Example 5).

In view of the many possible embodiments to which the principles of our invention may be applied, it should be recognized that the illustrated embodiment is only a preferred example of the invention and should not be taken as a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. An established human mast cell, wherein the cell expresses a functional FcεRI receptor.

2. The human cell of claim 1, wherein the cell exhibits cell growth enhanced by stem cell factor (SCF).

3. The human cell line of claim 1, wherein the cell expresses functional CD64 receptors.

4. An established human cell line that expresses functional FcεRI receptors and CD64 receptors, and wherein growth of the cell line is enhanced by stem cell factor (SCF).

5. The established human cell line of claim 4, wherein the cell line is LADl (ATCC No. PTA-3908) and mutants derived therefrom which retain a characteristic of the deposited cell.

6. The established human cell line of claim 4, wherein the cell line is LAD2 (ATCC No. PTA-3909) and mutants derived therefrom which retain a characteristic of the deposited cell.

7. The established human cell line of claim 4, wherein the cell line is LAD3 (ATCC No. PTA-3910) and mutants derived therefrom which retain a characteristic of the deposited cell.

8. The established human cell line of claim 4, wherein the cell line is LAD4 and mutants derived therefrom which retain a characteristic of the deposited cell.

9. The established human cell line of claim 4, wherein the cell line is LAD5 (ATCC No. PTA-3997) and mutants derived therefrom which retain a characteristic of the deposited cell.

10. A method for screening for an agent that affects mast cell proliferation, comprising contacting the established human mast cell of claim 1 with the agent; and measuring proliferation of the mast cell, thereby determining the effect of the agent on mast cell proliferation.

11. The method of claim 10, further comprising comparing the proliferation of the mast cell contacted with agent to a control.

12. The method of claim 11, wherein the control is an established mast cell not contacted with the agent.

13. The method of claim 6, wherein the agent is a polypeptide, pepidomimetic, or a chemical compound.

14. A method for screening for an agent that affects mast cell survival, comprising contacting the established human mast cell of claim 1 with the agent; and measuring survival of the mast cell, thereby determimng the effect of the agent on mast cell survival.

15. The method of claim 14, further comprising comparing the survival of the mast cell contacted with agent to a control.

16. The method of claim 15, wherein the control is an established mast cell not contacted with the agent.

17. The method of claim 14, wherein the agent is a polypeptide, pepidomimetic, or a chemical compound.

18. A method for screening for an agent that affects receptor cross linking, mediator release, or signal transduction in a mast cell, comprising contacting the established human mast cell of claim 1 with the agent; and measuring receptor cross linking, mediator release, or signal transduction in the mast cell, thereby determining the effect of the agent on receptor cross linking, mediator release, or signal transduction in the mast cell.

19. The method of claim 18, further comprising comparing the survival of the mast cell contacted with agent to a control.

20. The method of claim 19, wherein the control is an established mast cell not contacted with the agent.

21. The method of claim 18, wherein the agent is a polypeptide, pepidomimetic, or a chemical compound.

22. A kit for screening agents for their affect on a mast cell, comprising a container containing the established mast cell of claim 1.

23. The kit of claim 22, further comprising a containing comprising a medium or a factor essential for mast cell growth.

24. The kit of claim 22, further comprising written instructions.

25. A method for selecting an agent that affects human immunodeficiency virus infection, comprising contacting the established mast cell of claim 1 with the agent and the human immunodeficiency virus; and determining if the immunodeficiency virus infects the cell, thereby determining if the agent affects infection.

26. A method for selecting an agent that affects human immunodeficiency virus infection or replication, comprising, contacting a first established mast cell of claim 1 infected with a human immunodeficiency virus with the agent and with a second established mast cell of claim 1 not infected with the human immunodeficiency virus; and determining if the second established mast cell of claim 1 becomes infected with the human immunodeficiency virus; thereby determining if the agent affects HIN infection or replication.